EP0870040A2 - Ligands de ciblage de vehicules pour l'apport de genes - Google Patents

Ligands de ciblage de vehicules pour l'apport de genes

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Publication number
EP0870040A2
EP0870040A2 EP96945228A EP96945228A EP0870040A2 EP 0870040 A2 EP0870040 A2 EP 0870040A2 EP 96945228 A EP96945228 A EP 96945228A EP 96945228 A EP96945228 A EP 96945228A EP 0870040 A2 EP0870040 A2 EP 0870040A2
Authority
EP
European Patent Office
Prior art keywords
gene delivery
cells
vector
targeting
fusion protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96945228A
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German (de)
English (en)
Inventor
Sunil Chada
Theresa Banks
Margaret D. Moore
Stephen M. W. Chang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novartis Vaccines and Diagnostics Inc
Original Assignee
Chiron Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chiron Corp filed Critical Chiron Corp
Publication of EP0870040A2 publication Critical patent/EP0870040A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2881—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD71
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
    • C12N2710/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates generally to gene delivery vehicles, and more specifically, to methods for targeting gene delivery vehicles.
  • CD4 protein in-frame with the Avian Leukosis Virus transmembrane protein, or with the transmembrane protein of Murine Leukemia Virus, presumably in an attempt to target HIV infected T cells (Young et al. Science 250:1421, 1990). While the CD4 protein was expressed by the virus, no evidence was provided which showed that such viral particles were able to transduce target T cells.
  • the present invention provides recombinant gene delivery vehicles that are capable of targeting to cells bearing certain specific cell surface molecules or receptors.
  • the present invention provides these, as well as other related advantages.
  • the present invention provides compositions and methods for targeting gene delivery vehicles to a selected cell or tissue.
  • fusion proteins comprising a MHC Class I molecule, a MHC Class II molecule, or ⁇ 2 microglobulin and a targeting ligand.
  • the targeting ligand may be an antibody variable region, a hormone such as melanocyte stimulating hormone or erythropoietin.
  • fusion proteins comprising a MHC Class I molecule, a MHC Class II molecule, or ⁇ 2 microglobulin and one member of a high affinity binding pair.
  • the member of a high affinity binding pair is avidin.
  • nucleic acid molecule e.g., DNA, RNA, or some combination ofthe two
  • expression cassettes which are capable of directing the expression of such nucleic acid molecules, and host cells which contain these expression cassettes.
  • gene delivery vehicles which have on their surfaces one ofthe above-described fusion proteins.
  • Representative gene delivery vehicles include recombinant retroviruses and alphaviruses.
  • replication defective retroviral vector particles are provided which have a protein comprising heterologous MHC Class II molecule on its surface.
  • packaging cell lines comprising a gaglpol expression cassette, an env expression cassette, and an expression cassette which directs the expression of a sequence encoding one ofthe above-described fusion proteins.
  • vector producing cell lines which comprise such packaging cell lines and a recombinant retroviral vector.
  • methods for targeting a gene delivery vehicle to a selected cell type in a warm-blooded animal, comprising the step of administering to a warm-blooded animal one ofthe above-described gene delivery vehicles.
  • such methods comprise the general steps of (a) administering to a warm-blooded animal a gene delivery vehicle as described above, and (b) administering to the warm-blooded animal a targeting element coupled to a second member of a high affinity binding pair, the coupled targeting element being capable of specifically binding to a selected cell type in said warm-blooded animal, and the second member being capable of specifically binding to the first member, such that the gene delivery vehicle is targeted to the selected cell type.
  • the high affinity binding pair is selected from the group consisting of biotin/avidin, cystatin/papain, val-phosphonate/carboxy-peptidase A and 4CABP/RuBisCo.
  • the targeting element may be an antibody variable region or an immune accessory molecule.
  • the gene delivery vehicle may be a retroviral vector particle, or liposome or polycation condensed nucleic acids.
  • the gene delivery vehicle contains a heterologous sequence such as a gene encoding a cytotoxic protein (e.g., ricin, abrin, diphtheria toxin, cholera toxin, gelonin, pokeweed, antiviral protein, tritin, Shigella toxin and Pseudomonas exotoxin A), an antisense or ribozyme sequence, or an immune accessory molecule (e.g., IL-2, IL-12, IL-15, gamma-interferon, ICAM-1, ICAM-2, ⁇ - microglobin, LFA3, and HLA class I and HLA class II molecules).
  • a cytotoxic protein e.g., ricin, abrin, diphtheria toxin, cholera toxin, gelonin, pokeweed, antiviral protein, tritin, Shigella toxin and Pseudomonas exotoxin A
  • an antisense or ribozyme sequence
  • the heterologous sequence encodes a gene product that activates a compound with little or no cytotoxicity into a toxic product, such as, for example, HSVTK or VZVTK.
  • the heterologous sequence may be a replacement gene which encodes a protein such as Factor VIII, ADA, HPRT, CFTCR and the LDL Receptor.
  • the heterologous sequence may encode an immunogenic portion of a virus selected from the group consisting of HBV, HCV, HPV, EBV, FeLV, FIV and HIV.
  • Figure 1 is a schematic illustration of pSC6.
  • Figure 2 is a schematic illustration of pSC6/HLA-A2
  • Figure 3 provides a representative sequence of an HLA-A2 template.
  • Figure 4 provides a representative sequence of a gp350/220 peptide.
  • Figure 5 is a schematic illustration of pCRII/350-A2.
  • Figure 6 is a diagrammatic illustration ofthe location of PCR primers selected for amplification of a antibody variable region.
  • Figure 7 is a schematic illustration of pSC6/HLA-DR alpha.
  • Figure 8 provides a representative sequence of an HLA DR template.
  • Figure 9 provides a schematic illustration of pSC6 EPO- ⁇ 2 M
  • Gene delivery vehicle refers to a construct which is capable of delivering, and, within preferred embodiments expressing, one or more gene(s) or sequence(s) of interest in a host cell.
  • Representative examples of such vehicles include viral vectors, nucleic acid expression vectors, naked DNA, and certain eukaryotic cells (e.g., producer cells).
  • gene delivery vehicles ofthe present invention have a molecular weight of greater than about x kilodaltons, wherein x is selected from the group consisting of 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 4,000, and 5,000.
  • the gene delivery vehicle has on its surface a fusion protein (discussed below), either expressed on, or included as, an integral part ofthe exterior ofthe gene delivery vehicle.
  • Ve ⁇ tor construct refers to a nucleic acid construct which carries, and within certain embodiments, is capable of directing the expression of a nucleic acid molecule of interest.
  • the retroviral vector must include at least one transcriptional promoter/enhancer or locus defining element(s), or other elements which control gene expression by other means such as alternate splicing, nuclear RNA export, post-translational modification of messenger, or post-transcriptional modification of protein.
  • Such vector constructs must also include a packaging signal, long terminal repeats (LTRs) or portion thereof, and positive and negative strand primer binding sites appropriate to the retrovirus used (if these are not already present in the retroviral vector).
  • the recombinant retroviral vector may also include a signal which directs polyadenylation, selectable markers such as Neo, TK, hygromycin, phleomycin, histidinol, or DHFR, as well as one or more restriction sites and a translation termination sequence.
  • such vectors typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second strand DNA synthesis, and a 3' LTR or a portion thereof.
  • Retroviral gene delivery vehicle refers to a retrovirus which carries at least one gene of interest.
  • the retrovirus may also contain a selectable marker.
  • the recombinant retrovirus is capable of reverse transcribing its genetic material into DNA and inco ⁇ orating this genetic material into a host cell's DNA upon infection.
  • Nucleic acid expression vector or “Expression cassette” refers to an assembly which is capable of directing the expression of a sequence or gene of interest.
  • the nucleic acid expression vector must include a promoter which, when transcribed, is operably linked to the sequence(s) or gene(s) of interest, as well as a polyadenylation sequence.
  • the nucleic acid expression vectors described herein may be contained within a plasmid construct.
  • the plasmid construct may also include a bacterial origin of replication, one or more selectable markers, a signal which allows the plasmid construct to exist as single-stranded DNA (e.g., a Ml 3 origin of replication), a multiple cloning site, and a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication).
  • a bacterial origin of replication e.g., a Ml 3 origin of replication
  • a signal which allows the plasmid construct to exist as single-stranded DNA e.g., a Ml 3 origin of replication
  • a multiple cloning site e.g., a multiple cloning site
  • a "mammalian" origin of replication e.g., a SV40 or adenovirus origin of replication
  • Packaging cell refers to a cell which contains those elements necessary for production of infectious recombinant retrovirus which are lacking in a recombinant retroviral vector.
  • packaging cells typically contain one or more expression cassettes which are capable of expressing proteins which encode gag,pol and env proteins.
  • Producer cell or “Vector producing cell” refers to a cell which contains all elements necessary for production of recombinant retroviral vector particles.
  • High Affinity Binding Pair refers to a set a molecules which is capable of binding one another with a rj of less than 10" V M, wherein y is selected from the group consisting of 8, 9, 10, 11, 12, 13, 14 and 15.
  • KD refers to the disassociation constant ofthe reaction A + B ⁇ AB, wherein A and B are members ofthe high affinity binding pair.
  • affinity constants may be readily determined by a variety of techniques, including for example by a Scatchard analysis (see Scatchard, Ann. N. Y. Acad. Sci. 51:660-612, 1949).
  • suitable affinity binding pairs include biotin/avidin, cystatin/papain, phosphonate/ carboxypeptidase A, and 4CABP/RuBisCo.
  • “Targeting element” refers to a molecule which is capable of specifically binding a selected cell type.
  • targeting elements are considered to specifically bind a selected cell type when a biological effect of the coupled targeting element may be seen in that cell type, or, when there is greater than a 10 fold difference, and preferably greater than a 25, 50 or 100 fold difference between the binding ofthe coupled targeting element to target cells and non-target cells.
  • the targeting element bind to the selected cell type with a Kr j > of less than 10' ⁇ M, preferably less than 10" ⁇ M, more preferably less than 10"?M, and most preferably less than 10 " ⁇ M (as determined by a Scatchard analysis, see Scatchard, Ann. N. Y. Acad Sci. 51:660-672, 1949).
  • the targeting element bind to the selected cell type with an affinity of at least 1 log (i.e., 10 times) less than the affinity constant ofthe high affinity binding pair.
  • the Kr j value will be at least 1 log or 10 fold greater.
  • Suitable targeting elements are preferably non- immunogenic, not degraded by proteolysis, and not scavenged by the immune system.
  • Particularly preferred targeting elements (which are conjugated to a member ofthe high affinity binding pair) should have a half-life (in the absence of a clearing agent) within an animal of between 10 minutes and 1 week. Representative examples of suitable targeting elements are set forth below in more detail.
  • “Clearing agent” refers to molecules which can bind and/or cross-link circulating coupled targeting elements.
  • the clearing agent is non-immunogenic, specific to the coupled targeting element, and large enough to avoid rapid renal clearance.
  • the clearing agent is preferably not degraded by proteolysis, and not scavenged by the immune system.
  • Particularly preferred clearing agents for use within the present invention include those which bind to the coupled targeting element at a site other than the affinity binding member, and most preferably, which bind in a manner that blocks the binding ofthe targeting element to its target. Numerous cleaving agents may be utilized within the context ofthe present invention, including for example those described by Marshall et al. in Brit. J. Cancer 69:502-507, 1994.
  • compositions and methods for targeting a gene delivery vehicle to a selected cell type in a warm-blooded animal are based upon the discovery that fusion proteins which are composed of, for example, MHC Class I or Class II molecules, or ⁇ 2 microglobulin, and a targeting ligand or member of a high affinity binding pair, sort to the surface of a gene delivery vehicle during its construction.
  • fusion proteins which are composed of, for example, MHC Class I or Class II molecules, or ⁇ 2 microglobulin, and a targeting ligand or member of a high affinity binding pair, sort to the surface of a gene delivery vehicle during its construction.
  • fusion proteins which are composed of, for example, MHC Class I or Class II molecules, or ⁇ 2 microglobulin
  • a targeting ligand or member of a high affinity binding pair sort to the surface of a gene delivery vehicle during its construction.
  • such gene delivery vehicles may be utilized within the methods described herein in order to target the gene delivery vehicle to a specific or selected cell type, based upon the affinity of
  • non-virally encoded cell membrane molecules may be utilized for construction ofthe fusion protein described herein.
  • MHC Class I molecules, MHC Class II molecules, and ⁇ 2 microglobulin are provided as examples, that the present invention is not so limited.
  • non-virally encoded virion surface molecules may be utilized, including for example the transferrin receptor, CD43, CD44, CD63, CD71, adhesion molecules such as CD3, CD4, CD1 la, CD12, CD13, CD14, CD 15, CD16, CD17, CD18, LF1 (CD 11 a/CD 18), CD25, CD54, CD55 (DAF), CD59 (MIRL) or non ⁇ human animal equivalents (applies to all the markers).
  • transferrin receptor CD43, CD44, CD63, CD71
  • adhesion molecules such as CD3, CD4, CD1 la, CD12, CD13, CD14, CD 15, CD16, CD17, CD18, LF1 (CD 11 a/CD 18), CD25, CD54, CD55 (DAF), CD59 (MIRL) or non ⁇ human animal equivalents (applies to all the markers).
  • MHC major histocompatibility complex
  • MHC antigens proteins encoded by this complex
  • MHC class I and class II molecules both types of molecules are membrane- bound and present peptide antigens on the cell surface.
  • Class I molecules contain two separate polypeptide chains; an MHC encoded heavy chain (alpha chain) of approximately 44kD in humans and 47kD in mice, and a non- MHC encoded ⁇ -chain of 12kD.
  • the majority of the heavy chain extends extracellularly, with a short hydrophobic membrane spanning segment and a cytoplasmic carboxy terminal tail of approx. 30 amino-acids.
  • the ⁇ -chain interacts non-covalently with the extracellular portion of the heavy chain and is not directly attached to the cell surface.
  • the class I molecule can be segregated into four separate regions: the peptide-binding region
  • the Ig-like (alpha- 3) region of class I molecules links the peptide binding region to the transmembrane region. This region is highly conserved among all class I molecules examined and shares homology to Ig constant domains.
  • the ⁇ -chain of class I molecules is identical in all human class I molecules.
  • the class I ⁇ -chain is also known as B2-microglobulin, and like alpha-3 region, contains a disulphide linked loop homologous to an Ig constant domain. (Guessow et al., J. Immunol. 739:3132, 1987) (Robinson et al., Immunogenetics 20:655, 1984)
  • MHC class II molecules are composed of two polypeptide chains termed alpha chain (32-34 kD) and the beta chain (29-32 kD). Both class II chains are polymo ⁇ hic.
  • alpha chain 32-34 kD
  • beta chain 29-32 kD
  • Both class II chains are polymo ⁇ hic.
  • the three dimensional structure of Class II molecules has not yet been solved, however, primary sequence analysis reveals structural similarities between class I and class II molecules. Humans possess three class II gene loci encoding DR, DQ and DP molecules. Individual class II genes have previously been isolated and inserted into expression constructs by various investigators, including for example, DQw6A by Nishimura et al., J Immun.
  • fusion proteins ofthe present invention may include, in one aspect, one member of a high affinity binding pair.
  • high affinity binding pairs may be utilized, including for example, cystatin papain with an affinity of 10- 14 M (Bjork and Ylinenjarvi, Biochemistry 29: 1770-1776, 1990); val-phosponate/carboxypeptidase A with an affinity of 10" 1 M (Kaplan and Bartlett, Biochemistry 30:8165-8170, 1991); 4CABP-RuBisCo with an affinity of 10" 13 M, (Schloss, J. Biol. Chem.
  • a wide variety of other high affinity binding pairs may also be developed, for example, by preparing and selecting antibodies which recognize a selected antigen, and by further screening of such antibodies in order to select those with a high affinity (see generally, U.S. Patent Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993; see also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988).
  • antibodies or antibody fragments may also be produced and selected utilizing recombinant techniques (see William D.
  • targeting elements may be utilized within the context of the present invention, in order to specifically direct a gene delivery vehicle to a selected cell type.
  • targeting elements are proteins or peptides, although other non- proteinaceous molecules may also function as targeting elements.
  • antibodies which include, for example, antibody variable regions
  • Representative examples include anti-CD34 antibodies (e.g., 12.8 (Andrews et al., Blood 67:842, 1986), and MylO (Civin et al., J. Immunol.
  • TAG72 monoclonal Ab CC49 and B72.3 (King et ai., J. Biochem. 257:317-23, 1992) to target colon and breast cells, and the carcinoembryonic antigen monoclonal antibody ZCE025 (Nap et al., Cane. Res. 52:2329-39, 1992) to target colon carcinoma cells.
  • Suitable targeting elements include hormones and hormone receptors.
  • Representative examples include follicle stimulating hormone and lutenizing hormone to ovary and testes cells, melanocyte stimulating hormone and epidermal growth factor to epidermal cells, and human growth hormone to mostly bone cells and skeletal muscle cells.
  • immune accessory molecules may be utilized to target specific receptors on various cells. Examples include interferon targeted to macrophages and natural killer cells, interleukins to T-lymphocytes, and erythropoietin and CSF to bone marrow cells.
  • peptides such as substance P may target neurons as a mediator of pain signals, neuromedin (Conlon, J. Neurochem. 57:988, 1988) may be utilized to target the cells of the uterus for contractile activity and proteins corresponding to ligands for known cell surface receptors such as insulin may be utilized to target insulin receptors on cells for glucose regulation.
  • ligands and antibodies may be utilized to target selected cell types, including for example: monoclonal antibody c-SF-25 to target a 125kD antigen on human lung carcinoma (Takahashi et al, Science 259:1460, 1993); antibodies to various lung cancer antigens (Souhami, Thorax 47:53-56, 1992); antibodies to human ovarian cancer antigen HCl (Gallagher et al., Br. J. Cancer 64:35-40, 1991); antibodies to H/Le v /Leb antigens to target lung carcinoma (Masayuki et al., N. Eng. J. Med.
  • ligands may be selected from libraries created utilizing recombinant techniques (Scott and Smith, Science 249:386, 1990; Devlin et al., Science 249:404, 1990; Houghten et al., Nature 354:84 1991; Matthews and Wells, Science 260: ⁇ 113,1993; ⁇ issim et al., EMBOJ. 73(3):692-698, 1994), or equivalent techniques utilizing organic compound libraries (e.g., Eric Erb et al., Proc Natl Acad Sci. USA 97:1 1422-11426, 1994, K.S. Lam et al., Nature 354:82-84, 1991).
  • the targeting element or ligand may be a carbohydrate or other non-peptide component, that naturally modifies a particular type of protein sequence. Representative examples include sequences that allow addition of galactose terminal carbohydrates that then would allow targeting to the asyaloglycoprotein receptor on hepatocytes.
  • the targeting element may be added later by chemical or enzymatic methods and this may be facilitated by providing suitable peptide sequences in the hybrid molecule that favor the chemical or biochemical reaction required to add suitable ligand.
  • the targeting ligand may be a chimeric or fusion protein composed of two or more different proteins or non-protein components.
  • a protein generally assumes a specific three dimensional structure encoded by its amino acid sequence with a hydrophobic core and a more hydrophilic surface with a defined secondary, tertiary and often quaternary structure.
  • oligopeptide targeting ligands may be made from portions of a protein sequence that will not assume the full folded structure but may have a portion of the characteristic secondary structure and a characteristic amino acid sequence that defines a particular binding motif.
  • binding motifs are generally found in the surface-exposed regions ofthe protein, such as loops which are found between secondary structure units such as alpha helices or strands of beta sheet. Loops are generally flexible and can be highly variable in length and generally have few constraints on permissible sequence. Other binding regions of proteins may be found in clefts between domains of a single protein, or in regions where different elements of tertiary structure meet.
  • a new sequence in order to add a binding domain to another protein or to change a protein so that it will bind a specified target, a new sequence must be grafted into the existing one so as not to disrupt its structural integrity to such an extent that it cannot fold or be processed and transported properly in the cell.
  • the commonest site of insertion is at the amino or carboxyl end, leaving the rest ofthe protein intact.
  • Other sites of insertion of new sequence may involve replacement of entire domains of a polypeptide with another domain, such as in the case ofthe immunoadhesins, where IL-2, for example may replace the variable region of an intact immunoglobulin to make a hybrid molecule.
  • Small peptide sequences may also be inserted internally in proteins particularly at surface-exposed loops or in places where amino acid sequence alignments indicate that a high degree of sequence variability is tolerated or at the amino or carboxy termini.
  • Retroviral gene delivery vehicles ofthe present invention may be readily constructed from a wide variety of retroviruses, including for example, B, C, and D type retroviruses as well as spumaviruses and lentiviruses (see RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985).
  • retroviruses may be readily utilized in order to assemble or construct retroviral gene delivery vehicles given the disclosure provided herein, and standard recombinant techniques (e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Kunkle, PNAS 52:488, 1985).
  • portions ofthe retroviral gene delivery vehicles may be derived from different retroviruses.
  • retrovector LTRs may be derived from a Murine Sarcoma Virus, a tRNA binding site from a Rous Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Virus.
  • retroviral gene delivery vehicles that may be utilized within the context ofthe present invention include, for example, those described in EP 0,415,731; WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Patent No. 5,219,740; WO 93/1 1230; WO 93/10218; Vile and Hart, Cancer Res. 53:3860-3864, 1993; Vile and Hart, Cancer Res. 53:962-967, 1993; Ram et al., Cancer Res. 53:83-88, 1993; Takamiya et al., J. Neurosci. Res. 33:493-503, 1992; Baba et al., J.
  • recombinant retroviruses include those described in WO 91/02805 and WO 95/05789.
  • Packaging cell lines suitable for use with the above-described vector constructs may be readily prepared (see WO 92/05266), and utilized to create producer cell lines (also termed vector cell lines or "VCLs") for the production of recombinant vector particles, given the disclosure provided herein.
  • the present invention also provides a variety of Alphavirus vectors which may function as gene delivery vehicles.
  • Alphavirus vector systems may be constructed and utilized within the present invention. Representative examples of such systems include those described within WO 95/07994.
  • viral vectors systems may also be utilized as a gene delivery vehicle.
  • Representative examples of such gene delivery vehicles include viruses such as pox viruses, such as canary pox virus or vaccinia virus (Fisher-Hoch et al., PNAS 56:317-321, 1989; Flexner et al., Ann.
  • viral carriers may be homologous, non- pathogenic(defective), replication competent virus (e.g., Overbaugh et al., Science 239:906-910,1988), and nevertheless induce cellular immune responses, including CTL.
  • non-viral gene delivery vehicles may likewise be utilized within the context of the present invention.
  • Representative examples of such gene delivery vehicles include direct delivery of nucleic acid expression vectors, naked DNA alone (WO 90/11092), polycation condensed DNA linked or unlinked to killed adenovirus (Curiel et al., Hum. Gene Ther. 3:147-154, 1992), DNA ligand linked to a ligand with or without one ofthe high affinity pairs described above (Wu et al., J. of Biol. Chem 264:16985-16987, 1989), nucleic acid containing liposomes (e.g., WO 95/24929 and WO 95/12387) and certain eukaryotic cells (e.g., producer cells)
  • eukaryotic cells e.g., producer cells
  • nucleic acid molecules may be carried and/or expressed by the expression vectors or recombinant retroviruses ofthe present invention.
  • the nucleic acid molecules which are described herein do not occur naturally in the expression vector or recombinant retrovirus that carries it, and provides some desirable benefit, typically an ability to fight a disease, or other pathogenic agent or condition.
  • Substances which may be encoded by the nucleic acid molecules described herein include proteins (e.g., antibodies including single chain molecules), immunostimulatory molecules (such as antigens) immunosuppressive molecules, blocking agents, palliatives (such as toxins, antisense ribonucleic acids, ribozymes, enzymes, and other material capable of inhibiting a function of a pathogenic agent) cytokines, various polypeptides or peptide hormones, their agonists or antagonists, where these hormones can be derived from tissues such as the pituitary, hypothalamus, kidney, endothelial cells, liver, pancreas, bone, hemopoetic marrow, and adrenal.
  • proteins e.g., antibodies including single chain molecules
  • immunostimulatory molecules such as antigens
  • immunosuppressive molecules blocking agents
  • palliatives such as toxins, antisense ribonucleic acids, ribozymes, enzymes, and other material capable of inhibiting a
  • Such polypeptides can be used for induction of growth, regression of tissue, suppression of immune responses, apoptosis, gene expression, blocking receptor-ligand interaction, immune responses and can be treatment for certain anemias, diabetes, infections, high blood pressure, abnormal blood chemistry or chemistries (e.g., elevated blood cholesterol, deficiency of blood clotting factors, elevated LDL with lowered HDL), levels of Alzheimer associated amyloid protein, bone erosion/calcium deposition, and controlling levels of various metabolites such as steroid hormones, purines, and pyrimidines.
  • methods are provided for administration of an expression vector or a recombinant retrovirus which directs the expression of a palliative.
  • palliatives that act directly to inhibit the growth of cells include toxins such as ricin (Lamb et al., Eur. J. Biochem. 745:265-270, 1985).
  • the expression vector or recombinant retrovirus directs the expression of a substance capable of activating an otherwise inactive precursor into an active inhibitor of a pathogenic agent, or a conditional toxic palliative, which are palliatives that are toxic for the cell expressing the pathogenic condition.
  • inactive precursors may be converted into active inhibitors of a pathogenic agent.
  • expression vectors or recombinant retroviruses which direct the expression of a gene product (e.g., a protein) such as He ⁇ es Simplex Virus Thymidine Kinase (HSVTK) or Varicella Zoster Virus Thymidine Kinase (VZVTK) which assists in metabolizing antiviral nucleoside analogues to their active form are therefore useful in activating nucleoside analogue precursors (e.g., AZT or ddC) into their active form.
  • nucleoside analogue precursors e.g., AZT or ddC
  • AZT or ddl therapy will thereby be more effective, allowing lower doses, less generalized toxicity, and higher potency against productive infection.
  • nucleoside analogues whose nucleotide triphosphate forms show selectivity for retroviral reverse transcriptase but, as a result ofthe substrate specificity of cellular nucleoside and nucleotide kinases are not phosphorylated, will be made more efficacious.
  • expression vectors and recombinant retroviruses which have a therapeutic effect by encoding one or more ribozymes (RNA enzymes) (Haseloff and Gerlach, Nature 334:585, 1989) which will cleave, and hence inactivate, RNA molecules corresponding to a pathogenic function (see also, Foster and Symons, Cell 45: 211-220, 1987; Haseloff and Gerlach, Nature 328: 596-600, 1988; Ruffner et al.,
  • RNA enzymes RNA enzymes
  • expression vectors and recombinant retroviruses comprising a biologically active nucleic acid molecule that is an antisense sequence (an antisense sequence may also be encoded by a nucleic acid sequence and then produced within a host cell via transcription).
  • the antisense sequence is selected from the group consisting of sequences which encode influenza virus, HIV, HSV, HPV, CMV, and HBV.
  • the antisense sequence may also be an antisense RNA complementary to RNA sequences necessary for pathogenicity.
  • the biologically active nucleic acid molecule may be a sense RNA (or DNA) complementary to RNA sequences necessary for pathogenicity.
  • Still further aspects ofthe present invention relate to recombinant retroviruses capable of immunostimulation.
  • the ability to recognize and defend against foreign pathogens is essential to the function ofthe immune system.
  • the immune system must be capable of distinguishing "self from "nonself ' (i.e., foreign), so that the defensive mechanisms ofthe host are directed toward invading entities instead of against host tissues.
  • Cytolytic T lymphocytes are typically induced, or stimulated, by the display of a cell surface recognition structure, such as a processed, pathogen-specific peptide, in conjunction with a MHC class I or class II cell surface protein.
  • the invention provides methods for stimulating a specific immune response and inhibiting viral spread by using an expression vector or recombinant retroviruses that direct the expression of an antigen or modified form thereof in susceptible target cells, wherein the antigen is capable of either (1) initiating an immune response to the viral antigen or (2) preventing the viral spread by occupying cellular receptors required for viral interactions.
  • the expression vectors or recombinant retroviruses ofthe present invention may be constructed to express "immunomodulatory factors," many of which are set forth above.
  • Immunomodulatory factors refer to factors that, when manufactured by one or more ofthe cells involved in an immune response, or, which when added exogenously to the cells, causes the immune response to be different in quality or potency from that which would have occurred in the absence ofthe factor.
  • the quality or potency of a response may be measured by a variety of assays known to one of skill in the art including, for example, in vitro assays which measure cellular proliferation (e.g., 3 H thymidine uptake), and in vitro cytotoxic assays (e.g., which measure SlCr release) (see, Warner et al., AIDS Res. and Human Retroviruses 7:645-655, 1991). Immunomodulatory factors may be active both in vivo and ex vivo.
  • immunomodulatory factors include cytokines or lymhokines, interferons (e.g., ⁇ -IFN), tumor necrosis factors (TNFs) (Jayaraman et al., J. Immunology 744:942-951, 1990), CD3 (Krissanen et al., Immunogenetics 26:258-266, 1987), ICAM-1 (Airman et al., Nature 335:512-514, 1989; Simmons et al., Nature 337:624-627, 1988), ICAM-2, LFA-1, LFA-3 (Wallner et al., J. Exp. Med.
  • interferons e.g., ⁇ -IFN
  • TNFs tumor necrosis factors
  • CD3 Chirissanen et al., Immunogenetics 26:258-266, 1987
  • ICAM-1 Airman et al., Nature 335:512-514, 1989; Simmons et al., Nature 337:624-627, 1988
  • the present invention also includes expression vectors and recombinant retroviruses which encode immunogenic portions of desired antigens including, for example, viral, bacterial or parasite antigens.
  • hepatitis Band C viral antigens e.g., WO 93/15207
  • feline leukemia virus and/or immonudeficiency virus antigens e.g., WO 94/06921
  • Still other examples include expression vectors or recombinant retroviruses which direct the expression of a non- tumorigenic, altered genes such as the ras (ras*) gene and the p53 gene (see WO 93/10814).
  • the present invention provides gene delivery vehicles suitable for administration to humans and other warm-blooded animals.
  • a wide variety of methods may be utilized in order to produce recombinant viruses suitable for administration, including for example, the use of fermenters or bioreactors, roller bottles, cell hotels or cell factories, and hollow fiber culture.
  • cells are preferably grown on microcarriers (i.e., Cytodex 1 or Cytodex 2; Pharmacia, Piscataway, N.J. at concentrations ranging from 3 to 15 g/L microcarrier.
  • microcarriers i.e., Cytodex 1 or Cytodex 2; Pharmacia, Piscataway, N.J. at concentrations ranging from 3 to 15 g/L microcarrier.
  • suitable conditions include those described above for bioreactors, with the exception that microcarrier beads are generally not preferred. Representative examples of such methods, as well as other methods such as cell holds or cell factories and hollow linker culture, are described within WO 96/0926.
  • a wide variety of methods may be utilized for increasing viral concentration and purity, including for example, precipitation of recombinant retroviruses with ammonium sulfate, polyethylene glycol ("PEG”) concentration, concentration by centrifugation (either with or without gradients such as PERCOLL, or "cushions" such as sucrose, use of concentration filters (e.g., Amicon filtration), and 2- phase separations.
  • concentration filters e.g., Amicon filtration
  • the present invention provides several methods for the administration gene delivery vehicles.
  • methods are provided for targeting a gene delivery vehicle to a selected cell type in a warm-blooded animal, comprising the step of administering to a warm-blooded animal a gene delivery construct as described above which has a fusion protein containing a targeting ligand on its surface.
  • methods for targeting a gene delivery vehicle to a selected cell type in a warm-blooded animal, comprising the steps of (a) administering to a warm-blooded animal a gene delivery vehicle which has on its surface a fusion protein having one member of high affinity binding pair, and (b) administering to the animal a gene delivery vehicle coupled to a second member of said high affinity binding pair, the second member being capable of specifically binding to the first high affmity molecule such that the gene delivery vehicle is targeted to the selected cell type.
  • such methods further comprise, prior to the step of administering the gene delivery vehicle coupled to a second member of the high affinity binding pair, administering to the animal a clearing agent.
  • warm-blooded animals e.g. , humans, macaques, horses, cows, swine, sheep, dogs, cats, chickens, rats and mice
  • methods are also readily applicable to a variety of other animals, including, for example, fish.
  • gene delivery vehicles ofthe present invention may be administered to a wide variety of locations including, for example, into sites such as the cerebral spinal fluid, bone marrow, joints, arterial endothelial cells, rectum, buccal/sublingual, vagina, the lymph system, to an organ selected from the group consisting of lung, liver, spleen, skin, blood and brain, or to a site selected from the group consisting of tumors and interstitial spaces.
  • the gene delivery vehicles may be administered intraocularly, intranasally, sublinually, orally, topically, intravesically, intrathecally, topically, intravenously, intraperitoneally, intracranially, intramuscularly, or subcutaneously.
  • Other representative routes of administration include gastroscopy, ECRP and colonoscopy, which do not require full operating procedures and hospitalization, but may require the presence of medical personnel. The above-described methods may be readily utilized for a variety of therapeutic
  • the methods described above may be accomplished in order to inhibit or destroy a pathogenic agent in a warm-blooded animal.
  • pathogenic agents include not only foreign organisms such as parasites, bacteria, and viruses, but cells which are "foreign" to the host, such as cancer or tumor cells, or other cells which have been "altered".
  • the compositions described above may be utilized in order to directly treat pathogenic agents such as a tumor, for example, by direct injection into several different locations within the body of tumor.
  • arteries which serve a tumor may be identified, and the compositions injected into such an artery, in order to deliver the compositions directly into the tumor.
  • a tumor which has a necrotic center may be aspirated, and the compositions injected directly into the now empty center of the tumor.
  • the above-described compositions may be directly administered to the surface ofthe tumor, for example, by application of a topical pharmaceutical composition containing the retrovector construct, or preferably, a recombinant retroviral particle.
  • methods are provided for generating an immune response against an immunogenic portion of an antigen, in order to prevent or treat a disease, for suppressing graft rejection, for suppressing an immune response, and for suppressing an autoimmune response, utilizing the above-described compositions.
  • Retroviral backbones suitable for use with the present invention may be readily prepared by one of skill in the art. Representative examples of such backbones, such as KT-1 and KT-3, are described in WO 91/02805 and WO 95/05789.
  • a vector is first created in order to form the backbone for both the gag/pol and env expression cassettes. Briefly, pBluescript SK- phagemid (Stratagene, San Diego, Calif; GenBank accession number 52324; referred to as "SK-”) is digested with Spel and blunt ended with Klenow.
  • SK- pBluescript SK- phagemid
  • Dra I fragment of SV40 (Fiers et al., Nature 273:113- 120, 1978) from Dra I (bp 2366) to Dra I (bp2729) is then inserted into SK-, and a construct isolated in which the SV40 late polyadenylation signal is oriented opposite to the lacZ gene of SK-. This construct is designated SK-SV40A.
  • HCMV-IE Human Cytomegalovirus Major Immediate Early Promoter
  • the human HLA-A2 gene is isolated as a 4 kb Hind III - Apd X fragment (Koller and Orr, J. Immun. 734(4):2727-2733, 1985) and inserted into expression construct pSC6 (Invitrogen) using the Hind III site and the EcoR V sites in the polylinker of pSC6 ( Figure 1).
  • the expression construct contains the human CMV immediate early promoter driving the human HLA-A2 gene.
  • the ApaLl site is treated with T4 DNA polymerase to create a blunt end and the fragment is ligated to the pSC6 cut with Hind III and EcoR V ( Figure 2). This construct is designated pSC6/HLA-A2.
  • PCR primers are synthesized with the sequences specified in Table I in order to fuse the gp350/220 sequence to the HLA-A2 template at the mature amino terminus, and the PCR reactions are run as indicated in Table II according to the manufacturer's instructions with a GeneAmp PCR kit (Perkin/Elmer) ( Figures 3 and 4).
  • the PCR product containing the gp350/220 peptide sequence at the amino terminus of HLA-A2 is cloned into the vector pCRII according to the manufacturer's instructions using the TA cloning kit (Invitrogen, San Diego, CA) ( Figure 5).
  • the sequence of the PCR product is verified by standard DNA sequencing methods.
  • the fragment is removed from pCRII by digestion with the Eco47 III and BspEl sites and cloned into the pSC6/HLA-A2 vector ( Figure 2) at the Eco47 III and Bsp ⁇ sites to make the fusion vector, pSC6/350-A2.
  • the ecotropic envelope expression vector, pSC6/eco is created by inserting the Xbal-Nhel fragment of MoMLV (bp 5766 through bp 7845 of MoMLV) into pSC6 expression vector (Example 2A). Briefly the Xbal-Nhel envelope fragment is isolated from pMLV-K (Miller et al., J Vir. 49:214-222,1988) on an agarose gel. The fragment is then blunt-ended with T4 polymerase using standard methods, ligated into pSC6, digested at the EcoRV and Sm ⁇ l sites. The product in the correct orientation has a HCMV promoter followed by the complete ecotropic envelope coding sequence and an SV40 polyadenylation signal.
  • the ecotropic packaging cell line 293 ⁇ is made by cotransfecting 293 gag-pol ("293 2-3"; WO 91/02805; Burns, et al, PNAS 90: 8033-8037, 1993) with the pSC6/eco vector and pSV2gpt (Mulligan and Berg, Science 209:1422, 1980). After selection for gpt positive cells in the presence of selection medium, individual resistant colonies are isolated by dilution cloning and analyzed for expression by western blot analysis using goat polyclonal antibody for gp70 (NCI/BCB Repository Serum Number 79S000842, 1667 Davis St, Camden, NJ 08104).
  • gag/pol and MoMLV ecotropic envelope cell line, 293 E, or the gag/pol cell line 293 2-3 is co-transfected by CaPO4 precipitation (Wigler, M. et al., Cell 77:223, 1977) with 10 ⁇ g of pSC6/350-A2 and 1 ug ofthe phleomycin resistance vector, pUT507 (Mulsant et al., Somat. Cell Mol. Genet. 74:243-52, 1988).
  • pUT507 the phleomycin resistance vector
  • individual drug resistant cell colonies are expanded and analyzed for expression by western blot using monoclonal antibodies for HLA-A2.
  • the gene coding for thymidine kinase (ATP:thymidine 5' phosphotransferase, EC 2.7.1.21 ) of Herpes simplex virus type 1 is obtained from the plasmid p322TK (Enquist et al., Gene 7:335-342, 1979; Wagner et al., P.N.A. 75:1441-1445, 1981).
  • the promoter and the polyadenylation signal are removed and the gene is further modified to generate a 5' Xho I site and a 3' Cla I site. This modified gene fragment is then cloned into the Xho I and Cla I sites ofthe KT-3 backbone to generate the BH-1 vector.
  • the ATG methionine initiator codon for MoMLV gag expression has been mutated to ATT to inhibit gag expression (Chada et al., J. Vir. 67(6):3409-3417, 1993).
  • the MoMLV long terminal repeat (LTR) controls the expression ofthe HSV-tk gene and an internal S V40 promoter drives the expression of the neomycin resistance marker.
  • the retroviral packaging cell line, DA (Irwin et al., J. Vir. 65(8):5036-5044, 1994; Laube et al., Hum. Gene. Ther. 5:853-862, 1994), is engineered to express the murine leukemia virus gag/pol and amphotropic env sequences in the canine sarcoma cell line, D17.
  • the DA/N2/tk producer cell lines are generated by transduction of vesicular stomatitis virus G pseudotyped vector (Burns et al, P.N.A.S. 90:8033-8037, 1993) into the DA packaging cell line followed by selection in G418 (800 ⁇ g/ml).
  • clones are isolated by dilution cloning and tested for G418 ⁇ titer. The best producer clones (based on titer) DA/BH-1 #9A and #18 A are selected for subsequent use. These DA/N2/tk producer cells are grown to confluence, and supernatants containing the replication- defective N2/tk vectors are collected, filtered (pore size 0.45 um) and stored at -80°C. The vector titers are determined by serial dilution and CFU assay (Irwin et al., 1994) on HT- 1080 indicator cells selected in medium containing G418 (800 ug/ml). Titers are generally between 10 ⁇ and 10 ⁇ cfu/ml.
  • the DA/ ⁇ -gal producer cell line has been described previously (Irwin et al., 1994).
  • the packaging cell line 293E/350-A2 (Example 2E) is used to generate a producer cell line by transducing with vesicular stomatitis virus G pseudotyped vector (Burns et al, P.N.A.S. 90:8033-8037, 1993) into the 293E/350-A2 packaging cell line followed by selection in G418 (400 ⁇ g/ml). Individual clones are isolated by dilution cloning and tested for G418 ⁇ titer. The best producer clones (based on titer) are selected for subsequent use.
  • producer cells are grown to confluence, and supernatants containing the replication-defective N2/tk vectors are collected, filtered (pore size 0.45 um) and stored at -80°C.
  • the vector titers are determined by serial dilution (Irwin et al., 1994) on HT-1080 indicator cells selected in medium containing G418 (800 ug ml).
  • Vectors may be introduced into other cell lines as described above, and clones similarly selected.
  • This experiment uses the B lymphoblastoid cell line, Raji (ATCC CCL 56), which expresses the complement C3d receptor, CD21, as a target for transduction by the HLA fusion protein/ thymidine kinase vector and U937 (ATCC CRL 1593) a monocyte-like cell line (CD21 minus) as a control.
  • Two six well dishes are set up with 1 x 10 ⁇ of either Raji cells or U937 cells in five wells.
  • the wells are exposed to (1 ) thymidine kinase vector produced in an amphotropic packaging cell line, DA.
  • DA amphotropic packaging cell line
  • (3) thymidine kinase vector produced in the packaging cell line 293E, or 293 2-3 (4) irrelevant vector, DA/ ⁇ gal, (5) no vector.
  • Half of the cells are stained for thymidine kinase production and subjected to FACS analysis for production of thymidine kinase.
  • gancyclovir efficacy can be determined using relative levels of ⁇ H thymidine uptake (Bradley, L.M., in Selected Methods in Cellular Immunology, pp. 156- 161, (B. Mishell and S. Shiigi, eds.), Freeman & Co., N.Y. (1980).
  • Expression vectors encoding the different Class II genes are transfected into DA/ ⁇ gal cells using the CaPO4 procedure (described above) and transfected cells isolated by FACS sorting for cell surface expression ofthe appropriate Class II molecule (see below for details).
  • Initial experiments determine whether all three class II alleles can be expressed in canine DA cells. Pools of selected cells are expanded and ⁇ -gal (V) are isolated from confluent cultures.
  • HLA class II specific monoclonal antibodies are obtained from Pharmingen (San).
  • the antibodies are bound to magnetic beads (Dynal Inc., New Hyde Park, New York) according to the manufacturers instructions and used to purify ⁇ -gal(V) from the different producer cell lines. Vector bound to the beads is quantitated by ELISA using an anti-p30 detection antibody.
  • affinity columns may be prepared by binding the anti-class II monoclonal antibodies to sepharose using the amino-link kit according to the manufacturers instructions (Pierce, Rockford Illinois). Vector supernatants are passed over the columns and bound vector eluted using 1 M NaCl and titered for ⁇ -gal activity on HT 1080 cells. This assay can detect as little as one functional ⁇ -gal virion. Vector from the DA/ ⁇ -gal-DR cells is passed over the DR; DP and DQ columns and specificity of binding evaluated. Similar experiments are performed for vectors from control DA/ ⁇ gal cells as well as from DA/ ⁇ gal-DP and DA/ ⁇ gal-DQ cells. To assess preferential sorting of class II molecules to virion surfaces, DP, DQ and
  • DR are transfected into DA/ ⁇ gal cells and the resulting vector are tested as above for binding activity to class II specific columns.
  • variable regions of Mab 454A12 are cloned from the hybridoma cell line (ATCC HB 10804).
  • the heavy and light variable regions are obtained from the mRNA by PCR (Larrick et al., BioTechniques 7:934-838 (1989)) using mixed oligonucleotide primers that correspond to the leader sequences and the constant region sequences.
  • the products of the PCR are cloned into pCRII using the TA Cloning kit (Invitrogen) and sequenced by the Sanger dideoxynucleotide method. Three clones are sequenced in full to verify the DNA sequence of each V region. The sequences are given in Tables III and IV.
  • CHAIN Fv FRAGMENT The heavy and light chain variable regions are assembled to form a single chain Fv fragment with a synthetic linker sequence consisting of three repeats of Gly-Gly-Gly-Gly- Ser between them.
  • the primers, and templates for the assembly ofthe 454 A 12 sFv are summarized in Tables III through V.
  • the products ofthe two PCR reactions are used as templates in overlap PCR (Horton, R. M. et al., Biotechniques 5:528-535 (1990)) as shown in Table VI.
  • the overlap PCR product is then cloned into the vector pCRII via the TA cloning kit (Invitrogen) as previously described and corresponds to the single chain Fv fragment of approximately 750 bp in length.
  • TA cloning kit Invitrogen
  • Several clones are subjected to Sanger dideoxynucleotide sequencing to obtain a clone ofthe correct predicted DNA sequence.
  • the DR gene is inserted into the pSC6 vector at the EcoRV and EcoRI sites in the polylinker as a three piece ligation ofthe HLA DR gene cut from Sea I to Pst I (4 Kb fragment), and Pst I to EcoRI (1700 bp fragment). The Sea I creates a blunt end which ligates to the EcoR V site ofthe polylinker.
  • the exon containing the signal peptide and mature amino terminus is subjected to double overlap PCR from Nde I to Bgl II to add the sFv (described above section) at the amino terminus using primers described in Table VII and PCR reactions described in Table VIII.
  • the final product is cloned into pCRII for sequence verification.
  • the amino terminal Nde I to Bgl II fragment is cloned into the pSC6/HLA-DR to make pSC6/DR-aTfR.
  • gag/pol and MoMLV ecotropic envelope cell line, 293 E, or cell line 293 2-3 is co-transfected by CaPO4 precipitation (Wigler, M. et al., Cell 77:223, 1977) with 10 ⁇ g of pSC6/DR-aTfR and 1 ug ofthe phleomycin resistance vector, pUT507 (Mulsant et al., Somat. Cell Mol. Genet. 74:243-52, 1988).
  • the bacterial lacZ gene encoding ⁇ -galactosidase (" ⁇ gal”; Price, PNAS 54:156, 1987) is cloned into the N2 backbone as described (Irwin et al., 1994) to generate the N2 ⁇ gal provector plasmid.
  • N2 ⁇ gal provector is used to generate a stable producer cell clone termed DA/ ⁇ -gal. Purified vector supernatant has been generated from this cell line and concentrated to titers of greater than 1X10 ⁇ cfu ml.
  • the packaging cell line 293E/DR-aTfR or, 293 2-3/DR-aTfR, is used to generate a producer cell line by transducing with vesicular stomatitis virus G pseudotyped vector (Burns et al., P.N.A.S. 90:8033-8037, 1993) into the 293E/DR-aTfR packaging cell line, followed by selection in G418 (400 ⁇ g/ml). Individual clones are isolated by dilution cloning and tested for G418 ⁇ - titer.
  • the best producer clones are selected for subsequent use. These producer cells are grown to confluence, and supernatants containing the replication-defective N2- ⁇ Gal vectors are collected, filtered (pore size 0.45 um) and stored at -80°C. The vector titers are determined by serial dilution (Irwin et al., 1994) on HT-1080 indicator cells selected in medium containing G418 (800 ug/ml).
  • This experiment uses the cell line Molt4 (ATCC CRL 1582) which expresses the transferrin receptor as a target for transduction by the HLA DR-antiTfR/ ⁇ Gal vector.
  • two six well dishes are set up with 1 x 10 ⁇ of MOLT4 cells in four wells.
  • the wells are exposed to (1) ⁇ gal vector produced in the amphotropic packaging cell line, DA (Example 3E), (2) ⁇ gal vector produced in the HLA-A2 fusion vector packaging cell line,
  • Half of the cells are stained for ⁇ gal production.
  • the other half are subjected to treatment with 1000 ⁇ g/ml geneticin for 10 days and live cells counted (Irwin, et al., 1994, supra).
  • the basic strategy employed for the generation ofthe EPO-b2m fusion protein is to use PCR with the appropriate primers and templates to produce EPO and b2m fragments which can then be ligated with the appropriately digested eukaryotic expression vector pSC6.
  • a three way ligation is accomplished by utilizing a Cla -Aatl EPO fragment, an Aatl-EcoRl b2m fragment and a C/ ⁇ l-EcoRI- digested pSC6 expression vector.
  • the plasmid ⁇ POenv-D5923 (Kasahara et al., Science 266:1373, 1994) is utilized as the source ofthe human EPO gene.
  • This clone contains cDNA which encodes the complete mature EPO protein of 166 amino acids in length and which can be excised intact from the vector using the restriction enzymes BstE II and BamHI.
  • the BstE II site is treated with T7 polymerase to create a blunt end and the fragment is then ligated with EcoRV and BamHI digested plasmid SK+ (Stratagene, La Jolla, CA).
  • This plasmid construct is termed SK+/ ⁇ PO and the presence of a Clal site 5' to the EcoRV site in the muhicloning site of SK+ allows the ⁇ PO gene to be removed from the SK+/ ⁇ PO plasmid as a Clal-Aatl fragment.
  • the Aatl site is a unique restriction site located near the end of the 5th exon ofthe EPO gene and is located before the termination codon.
  • the human b2m gene fragment is engineered to contain an Aatl restriction site at its 5' end and an EcoRI restriction site at its 3' end.
  • the plasmid p714 (Parker and Wiley, Gene S3 : 1 17, 1989) containing cDN A encoding human b2m serves as the PCR template for a set of primers which will amplify a gene fragment containing the b2m gene (minus its signal peptide) as well as all the necessary sequences to fuse the 3' end ofthe ⁇ PO gene (minus its termination codon) in frame with the b2m gene.
  • the primer set has been designed as follows: Primer 1 : 5' GGGAGGCCTG CAGGACAGGG GACAGACAGC GTACTCCAAA GATTCAGGTT 3' (Seq. ID No. 17) and contains the Aatl restriction site, the last several codons ofthe ⁇ PO gene and the first several codons of the b2m gene.
  • Primer 2 is the antisense sequence containing the EcoRI restriction site and the last 9 codons ofthe b2m gene, (5' GGGAATTCTA CCTGGCGCTG TTACATGTCT CGGTC 3' (Seq. ID No. 18).
  • the PCR reaction is run according to manufacturer's instructions with a Gene Amp PCR kit (Perkin/ ⁇ lmer) and the resulting amplified gene product is cloned into the vector pCRII according to the manufacturer's instructions using the TA cloning kit (Invitrogen, San Diego, CA). The sequence ofthe amplified PCR product is verified using standard DNA sequencing methods. The fragment is then removed from pCRII by digestion first with Aatl and then by partial digestion with EcoRI (there is an additional EcoRI site 209 bp 5' from the terminal EcoRI which is located just before the termination codon ofthe b2m gene). Pilot digestions are set up to determine the optimal conditions for digesting the terminal EcoRI site while leaving the other EcoRI site undisturbed.
  • the correct Aatl -EcoRI b2m fragment is ligated simultaneously with the Clal-Aatl ⁇ PO gene fragment as well as with C/ ⁇ l-EcoRI digested pSC6 vector DNA.
  • the resulting fusion construct pSC6/ ⁇ PO-b2 is sequenced to verify that the EPO-b2m gene fusion is correct.
  • the gag/pol and MoMLV ecotropic envelope cell line, 293E (Example 2D) or 293 2-3 is co-transfected by CaPO4 precipitation (Wigler et al., Cell 77:223, 1977) with 10 mg of pSC6/EPO-b2m plasmid DNA and 1 ug ofthe phleomycin resistance vector, pUT507 (Mulsant et al., Somat. Cell Mol. Genet. 14:243-52, 1988).
  • pUT507 the phleomycin resistance vector
  • individual drug resistant cell colonies are expanded and analyzed for expression by western blotting and fluorescent staining using monoclonal antibodies for EPO and for human b2m.
  • cells expressing high levels of EPO-b2m may be selected using a Fluorescence Activated Cell Sorter (FACS).
  • FACS Fluorescence Activated Cell Sorter
  • EPO detection polyclonal antiserum to EPO (AB-286-NA; R & D Systems, Minneapolis, MN) are used and for b2m detection, mAb BBM-1, an antibody which binds an epitope present in both native and denatured b2m (Parham, et al., J. 73 o/. Chem. 258: 6179-6186, 1983) are used.
  • the packaging cell line 293E/EPO-b2m, or 293 2-3/EPO-b2m is used to generate a producer cell line by transducing the packaging cell line with vesicular stomatitis virus G pseudotyped vector (Burns et al., P.N.A.S. 90:8033-8037, 1993), followed by selection in G418 (400 ⁇ g/ml). Individual clones are isolated by dilution cloning and tested for
  • G418 ⁇ - titer The best producer clones (based on titer) are selected for subsequent use. These producer cells are grown to confluence, and supernatants containing the replication- defective N2- ⁇ Gal vectors are collected, filtered (pore size 0.45 um) and stored at -80°C The vector titers are determined by serial dilution (Irwin et al., 1994) on HT-1080 indicator cells selected in medium containing G418 (800 ug/ml). D. TARGETING TO EPO RECEPTORS.
  • This experiment uses a cell line in which NIH 3T3 cells are stably transfected with a complementary DNA (cDNA) encoding the EPO receptor (cDNA source is A. D' Andrea et al., Cell 57: 277, 1989). Standard transfection and selection methodologies are used to isolate NIH 3T3 cells stably expressing the EPO receptor (NIH 3T3-EPOR) and FACS analysis is used to confirm the expression of the EPO receptor on these cells. Two six well dishes are set up with 1 x 10 5 of the NIH 3T3-EPOR cells or NIH 3T3 cells (does not contain EPO receptor) in four wells.
  • cDNA complementary DNA
  • the wells are then exposed to (1) bgal vector produced in an amphotropic packaging cell line, DA, (2) ⁇ gal vector produced in the EPO- b2m fusion vector packaging cell line, (3) ⁇ gal vector produced in the packaging cell line 293E, (4) no vector.
  • Half of the cells are stained for bgal production. The other half are subjected to treatment with 600 ug/ml geneticin for 10 days and live cells counted (Irwin, et al., 1994).
  • the recombinant EPO-b2m fusion protein may be expressed using the prokaryotic expression vector pSE280 (Invitrogen, San Diego, CA).
  • the EPO-b2m gene is excised from pSC6/EPO-b2m as a CM-EcoRI fragment by performing the Clal digestion first, treating with klenow to generate a blunt end, followed by an EcoRI-partial digestion. This fragment is then ligated with NcoI-EcoRI digested pS ⁇ 280 vector D ⁇ A, (Ncol end is also blunted prior to setting up the ligation).
  • This recombinant plasmid is termed pSE280/EPO-b2m.
  • EXPRESSION OF EPQ-B2M FUSION PROTEIN FOR SCALE UP AND PURIFICATION Briefly, the pSE280 plasmid containing the EPO-b2m fusion protein (pSE280/EPO-b2m) is transformed into E. coli strain XA90 and positive protein- producing clones are then sequenced to verify their DNA sequence. Subsequently, positive clones are grown to the appropriate density and induced with isopropyl b-D- thiogalactopyranoside (IPTG;lmM).
  • IPTG;lmM isopropyl b-D- thiogalactopyranoside
  • the cultures are harvested (1 liter aliquots) by centrifugation and subsequently lysed using a solution of lOmM Tris-HCl, pH 8 (20 ml/1 liter of pelleted cells), containing lysozyme at 100 ug/ml, the protease inhibitor PMSF at 50 ug/ml, DNase at 20 ug/ml, RNase at 20 ug/ml, and 1 mM EDTA. Cells are incubated in this mixture for 20 min. at RT and then sonicated before another round of centrifugation at 10,000 x g for 20 min.
  • the resulting pellet containing the recombinant protein is washed with 20 ml of lOmM Tris-HCl, pH 8 and then resuspended in solution of lOOmM Tris-HCl, pH 8 and 8M urea (10ml total). Following resuspension, the lysate is centrifuged at 150,000 x g for lhr at 4°C. The recombinant fusion protein in urea is then dialyzed against 10 mM Tris-HCl, pH7, and purified on Q Sepharose in lOmM Tris-HCl, pH7, with a linear gradient from 0-100 mM NaCl. Fractions are collected and concentrated by ultrafiltration using Centricon filters (Amicon).
  • 5 x 10 4 cpm/ng 5 x 10 4 cpm/ng. Briefly, 5 x 10 5 HT1080 cells in 1 ml of RPMI medium containing gelatin at 1 mg/ml and puromycin at 50 ug/ml are incubated with 20 ug of labeled b2m for
  • HBSS Hanks buffered saline
  • Immunoprecipitation reactions use the BBM-1 monoclonal antibody which is directed against an epitope present in both native and denatured b2m. After immunoprecipitation ofthe labeled cells, the radioactive label is detected indicating that the labeled b2m had been exchanged with the native b2m associated with these cells. Purified EPO-b2m fusion protein is exchanged with the b2m present on the vector surface as described above. Following the exchange reaction, this "exchanged" vector is tested for targeting to cells expressing the EPO receptor, (as described in example 4D). Amicon filtration (300k molecular weight cutoff) is used to purify the vector from contaminating substances.
  • Two six well dishes are set up with 1 x 10 of the NIH 3T3-EPOR cells or NIH 3T3 cells (does not contain EPO receptor) in four wells.
  • the wells are then exposed to (1) ⁇ gal vector produced in an amphotropic packaging cell line, DA, (2) ⁇ gal vector produced as in (1 ) but following the EPO-b2m fusion protein exchange, (3) ⁇ gal vector produced in the packaging cell line 293 E, (4) no vector.
  • Half of the cells are stained for bgal production. The other half are subjected to treatment with 600 ug/ml geneticin for 10 days and live cells counted (Irwin et al., 1994).
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • ATGTTCCTCA GCAGCCTGGC CTCTGAGGAC TCTGCGATCT ATTTCTGTGC AAGAGATACT 300
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

La présente invention concerne des protéines fusionnées constituées par une molécule CMH de classe I ou II ou une microglobuline β2; l'invention concerne aussi un ligand de ciblage. L'invention concerne, de plus, des molécules d'acide nucléique qui codent de telles protéines fusionnées ainsi que des cassettes d'expression appropriées et des cellules hôtes. Elle concerne également des procédés de ciblage d'un véhicule d'apport de gènes sur un type de cellule sélectionné, au moyen de véhicules d'apport de gènes qui contiennent, sur leurs surfaces, l'une des protéines fusionnées précitées.
EP96945228A 1995-12-29 1996-12-20 Ligands de ciblage de vehicules pour l'apport de genes Withdrawn EP0870040A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US941195P 1995-12-29 1995-12-29
US58054195A 1995-12-29 1995-12-29
US580541 1995-12-29
US9411P 1995-12-29
PCT/US1996/020295 WO1997024446A2 (fr) 1995-12-29 1996-12-20 Ligands de ciblage de vehicules pour l'apport de genes

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US6555651B2 (en) * 1997-10-09 2003-04-29 The Trustees Of Columbia University In The City Of New York Ligand binding site of rage and uses thereof
CA2326407C (fr) * 1998-04-29 2012-09-11 University Of Southern California Vecteurs retroviraux incluant des proteines d'escorte a enveloppe modifiee
US7078483B2 (en) 1998-04-29 2006-07-18 University Of Southern California Retroviral vectors including modified envelope escort proteins
US7521197B2 (en) 1998-06-05 2009-04-21 Alexis Biotech Limited Method for producing cytotoxic T-cells
US7264965B2 (en) 1998-06-05 2007-09-04 Alexis Biotech Limited Method for producing or enhancing a T-cell response against a target cell using a complex comprising an HLA class I molecule and an attaching means
GB2339782A (en) * 1998-06-05 2000-02-09 Philip Michael Savage Chimeric protein complexes comprising HLA class I antigens
US6682741B1 (en) * 1998-06-10 2004-01-27 The United States Of America As Represented By The Department Of Health And Human Services β2 microglobulin fusion proteins and high affinity variants
BR0008645A (pt) * 1999-03-03 2002-01-22 Univ Pennsylvania Métodos de induzir uma resposta imunológica contra um imunógeno em um indivìduo, de reduzir rejeição de células, tecido ou órgão doadores nã0-assemelhados em um indivìduo e uma resposta imunológica dominante em um indivìduo e de expandir uma subpopulação de células t associadas com uma resposta imunológica especìfica, plasmìdeo, e, composição
US20040191260A1 (en) 2003-03-26 2004-09-30 Technion Research & Development Foundation Ltd. Compositions capable of specifically binding particular human antigen presenting molecule/pathogen-derived antigen complexes and uses thereof
AU2008243241B2 (en) * 2001-06-19 2011-11-17 Technion Research And Development Foundation Ltd. Methods and pharmaceutical compositions for immune deception, particularly useful in the treatment of cancer
US20030017134A1 (en) * 2001-06-19 2003-01-23 Technion Research And Development Foundation Ltd. Methods and pharmaceutical compositions for immune deception, particularly useful in the treatment of cancer
US8022190B2 (en) 2001-06-19 2011-09-20 Technion Research & Development Foundation Ltd. Immuno-molecules containing viral proteins, compositions thereof and methods of using
EP2024507B1 (fr) 2006-05-19 2015-07-22 Technion Research and Development Foundation Ltd. Protéines de fusion, leurs utilisations et leurs procédés de production

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DE3825615A1 (de) * 1988-07-28 1990-02-01 Behringwerke Ag Antigenkonstrukte von "major histocompatibility complex" klasse i antigenen mit spezifischen traegermolekuelen, ihre herstellung und verwendung
GB9307371D0 (en) * 1993-04-08 1993-06-02 Walls Alan J Fusion proteins
PT758394E (pt) * 1994-05-02 2003-04-30 Bernd Groner Proteina bifuncional sua preparacao e utilizacao
ATE200105T1 (de) * 1994-05-13 2001-04-15 Chiron Corp Verfahren und zusammensetzungen als vehikel zur zielgerichtete einbringen von genen

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